The present disclosure relates generally to assays and methods for identifying inhibitors for the treatment of cancers. More particularly, the present disclosure relates to screening assays and methods for identifying neddylation inhibitors for treating cancers, tumor suppressor activators in a cancer, and identifying inhibitors of tumor-induced angiogenesis.
c-Src is one of the nine members of the Src-family kinases. c-Src is a cytoplasmic non-receptor tyrosine kinase that is a critical initiation site for multiple signal transduction pathways and interactions. c-Src activation has been implicated in a multitude of tumor progression properties including survival, apoptosis, angiogenesis, migration, and adhesion. While c-Src is rarely mutated in human cancers, aberrant activation is correlated with a clinical progression of cancer and high levels are seen in a number of human tumors. However, tumorigenesis is usually in coordination with another oncogene, such as Her2 in breast cancer, since c-Src alone is not a strong transforming agent.
One of the downstream effectors of c-Src is the survival factor Akt. Akt has been shown to phosphorylate many substrates to promote cell survival including the murine double minute-2 protein (Mdm2) Mdm2 undergoes nuclear translocation in response to Akt phosphorylation at serines 166 and 186. Once in the nucleus, Mdm2 binds to the tumor suppressor p53 and inhibits its transcriptional activity along with functioning as an E3 ubiquitin ligase to signal nuclear export and proteasomal degradation. While normally kept at low levels, p53 is stabilized through post-translational modifications to both itself and Mdm2 in response to genotoxic stress. Mdm2 can also regulate p53 function by conjugation of Nedd8. This modification results in a stable, but a transcriptionally inactive p53.
Nedd8 is a part of the ubiquitin-like protein family. In a similar fashion to the ubiquitin system, neddylation involves the activation and transfer of Nedd8 from E1 (APP-BP1 in human), E2 (Ubc12), and multiple E3 RING ligases and can be removed by de-neddylating enzymes, such as COPS, CSN, NEDP1. The Nedd8 pathway has been demonstrated to be important for viability in mice, C. elegans, and S. pombe. In addition, the mammalian cell line TS-41, which has a temperature sensitive mutation in the SMC gene (APP-BP1 in human), participates in multiple rounds of DNA replication without entering a mitotic cycle. Nedd8 conjugation has been shown to be upregulated in certain types of cancer and recently a general inhibitor of the Nedd8 pathway, MLN4924, has been developed. Inhibition of global neddylation leads to apoptotic death in human cancer cells and suppresses the growth of human tumors in mouse xenografts.
In the tumor microenvironment, there are numerous growth factors and cytokines secreted from various invading cells and the stroma. This signaling leads to p53 levels that are generally not lower, but elevated. This suggests that the Mdm2-mediated destabilization of p53 was not functional. The present disclosure is directed to a Mdm2/c-Src pathway leading to activation of Mdm2 neddylation. Accordingly, Mdm2 activity presents an alternative mechanism for identifying targeted cancer therapies.